Air liquid interface cell culture (ALI) is a method of cell culture by which basal stem cells are grown with their basal surfaces in contact with media, and the top of the cellular layer is exposed to the air. The cells are then lifted and media is changed until the development of a mucociliary phenotype of a pseudostratified epithelium, similar to the tracheal epithelium.[1][2]
Pseudostratified epithelium of the trachea ALI cell culture aims to recreate in vitro
This method of cell culture aims to be used to study fundamental aspects of the respiratory epithelium, such as cell-to-cell signaling, disease modeling, and respiratory regeneration.[1][2]
Air-liquid interface cell culture compares to standard cell culture practices by specifically aiming to restore the pseudostratified striation of the respiratory airway in vitro, and aiming to maintain the respiratory airway-niche of (from top to bottom) 1) air, 2) pseudostratified epithelium, and 3) liquid media. Standard cell culture processes are either non-airway specific or revolve around an organ system that requires other means of cellular maintenance in vitro.
The protocol for air-liquid interface culture relies on two key steps: isolation of the epithelial cells from an organism, and culture of those cells.
Isolationedit
Organs with epithelial cells are isolated[1] (dissected) and placed in PBS to clean any impurities and these isolated organs are digested using trypsin. After the digest, ROCK inhibitor (Rho-associated kinase inhibitor) is added to prevent cellular apoptosis of isolated cells. What is left is digested cellular content mixed with tissue; pronase/DNAse is added to clean up dead cellular material as well as any remaining protein material. The resulting material is chopped finely in media and individual isolated cell samples are placed in respectively marked microcentrifuge tubes, and shaken at 37 °C for 30 – 40 minutes. Once the cells have been incubated, they are plated onto transwell membranes for growth.
Cultureedit
Plate cells with SABM (Small Airway Basal Media) to enrich the cells with nutrients for growth and eventual differentiation.[1] Once the cells are plated, they are grown for 3–7 days under careful observation (while changing media each day) until desired confluence is reached.
More recently, a study on In vitro generation of type-II pneumocytes initiated from human CD34(+) stem cells has been demonstrated the air-liquid interface cell culture method precisely.[3]
Uses in scientific studyedit
Air-liquid interface cultures are used in many stem cell studies that aim to recreate a pseudostratified epithelium in vitro. These studies can contribute to new findings on various topics:
Canceredit
In modeling cancer and various other diseases, the stem cells in the basal layer of the tracheal epithelium (basal stem cells) were isolated and used in developing 3D organoids that could be used for various studies, including tumor studies. The cell culture method used involves the isolation of cells into culturing with growth factors to grow over time. Once the cells had been grown, they were mixed with Matrigel and cultured to form 3D organoids.[4][5]
Cell growth/differentiationedit
In modeling the pseudostratified epithelium in vitro, more cellular studies have been performed in order to determine the nature of the cells – their differentiation pathways, their growth mechanism, and their repair/response mechanism in the state of post-injury. One recent study has shown that differentiated cells in the respiratory epithelium (secretory cells and ciliated cells primarily) can dedifferentiate into their naive status, and become stem-like again.[1][5]
Adapted protocol: maintenance of stem-like properties in basal stem cellsedit
As an adaptation to the air-liquid interface culture protocol, auxiliary protocols have been developed using the ALI framework to maintain naive stem cell properties for extensive periods of time.[6]
^ ab"Air-Liquid Interface Culture for Respiratory Research". www.stemcell.com. Retrieved 2018-03-30.
^Srikanth, Lokanathan; Venkatesh, Katari; Sunitha, Manne Mudhu; Kumar, Pasupuleti Santhosh; Chandrasekhar, Chodimella; Vengamma, Bhuma; Sarma, Potukuchi Venkata Gurunadha Krishna (February 2016). "In vitro generation of type-II pneumocytes can be initiated in human CD34(+) stem cells". Biotechnology Letters. 38 (2): 237–242. doi:10.1007/s10529-015-1974-2. ISSN 1573-6776. PMID 26475269. S2CID 17083137.
^Rock, Jason R.; Onaitis, Mark W.; Rawlins, Emma L.; Lu, Yun; Clark, Cheryl P.; Xue, Yan; Randell, Scott H.; Hogan, Brigid L. M. (2009-08-04). "Basal cells as stem cells of the mouse trachea and human airway epithelium". Proceedings of the National Academy of Sciences. 106 (31): 12771–12775. Bibcode:2009PNAS..10612771R. doi:10.1073/pnas.0906850106. ISSN 0027-8424. PMC2714281. PMID 19625615.
^ abTata, Purushothama Rao; Rajagopal, Jayaraj (2017-03-01). "Plasticity in the lung: making and breaking cell identity". Development. 144 (5): 755–766. doi:10.1242/dev.143784. ISSN 0950-1991. PMC5374348. PMID 28246210.
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Air liquid interface cell culture ALI is a method of cell culture by which basal stem cells are grown with their basal surfaces in contact with media and the top of the cellular layer is exposed to the air The cells are then lifted and media is changed until the development of a mucociliary phenotype of a pseudostratified epithelium similar to the tracheal epithelium 1 2 Pseudostratified epithelium of the trachea ALI cell culture aims to recreate in vitroThis method of cell culture aims to be used to study fundamental aspects of the respiratory epithelium such as cell to cell signaling disease modeling and respiratory regeneration 1 2 Air liquid interface cell culture compares to standard cell culture practices by specifically aiming to restore the pseudostratified striation of the respiratory airway in vitro and aiming to maintain the respiratory airway niche of from top to bottom 1 air 2 pseudostratified epithelium and 3 liquid media Standard cell culture processes are either non airway specific or revolve around an organ system that requires other means of cellular maintenance in vitro Contents 1 Protocol 1 1 Isolation 1 2 Culture 2 Uses in scientific study 2 1 Cancer 2 2 Cell growth differentiation 3 Adapted protocol maintenance of stem like properties in basal stem cells 4 ReferencesProtocol editThe protocol for air liquid interface culture relies on two key steps isolation of the epithelial cells from an organism and culture of those cells Isolation edit Organs with epithelial cells are isolated 1 dissected and placed in PBS to clean any impurities and these isolated organs are digested using trypsin After the digest ROCK inhibitor Rho associated kinase inhibitor is added to prevent cellular apoptosis of isolated cells What is left is digested cellular content mixed with tissue pronase DNAse is added to clean up dead cellular material as well as any remaining protein material The resulting material is chopped finely in media and individual isolated cell samples are placed in respectively marked microcentrifuge tubes and shaken at 37 C for 30 40 minutes Once the cells have been incubated they are plated onto transwell membranes for growth Culture edit Plate cells with SABM Small Airway Basal Media to enrich the cells with nutrients for growth and eventual differentiation 1 Once the cells are plated they are grown for 3 7 days under careful observation while changing media each day until desired confluence is reached More recently a study on In vitro generation of type II pneumocytes initiated from human CD34 stem cells has been demonstrated the air liquid interface cell culture method precisely 3 Uses in scientific study editAir liquid interface cultures are used in many stem cell studies that aim to recreate a pseudostratified epithelium in vitro These studies can contribute to new findings on various topics Cancer edit In modeling cancer and various other diseases the stem cells in the basal layer of the tracheal epithelium basal stem cells were isolated and used in developing 3D organoids that could be used for various studies including tumor studies The cell culture method used involves the isolation of cells into culturing with growth factors to grow over time Once the cells had been grown they were mixed with Matrigel and cultured to form 3D organoids 4 5 Cell growth differentiation edit In modeling the pseudostratified epithelium in vitro more cellular studies have been performed in order to determine the nature of the cells their differentiation pathways their growth mechanism and their repair response mechanism in the state of post injury One recent study has shown that differentiated cells in the respiratory epithelium secretory cells and ciliated cells primarily can dedifferentiate into their naive status and become stem like again 1 5 Adapted protocol maintenance of stem like properties in basal stem cells editAs an adaptation to the air liquid interface culture protocol auxiliary protocols have been developed using the ALI framework to maintain naive stem cell properties for extensive periods of time 6 References edit a b c d e Tata Purushothama Rao Mou Hongmei Pardo Saganta Ana Zhao Rui Prabhu Mythili Law Brandon M Vinarsky Vladimir Cho Josalyn L Breton Sylvie November 2013 Dedifferentiation of committed epithelial cells into stem cells in vivo Nature 503 7475 218 223 Bibcode 2013Natur 503 218T doi 10 1038 nature12777 ISSN 1476 4687 PMC 4035230 PMID 24196716 a b Air Liquid Interface Culture for Respiratory Research www stemcell com Retrieved 2018 03 30 Srikanth Lokanathan Venkatesh Katari Sunitha Manne Mudhu Kumar Pasupuleti Santhosh Chandrasekhar Chodimella Vengamma Bhuma Sarma Potukuchi Venkata Gurunadha Krishna February 2016 In vitro generation of type II pneumocytes can be initiated in human CD34 stem cells Biotechnology Letters 38 2 237 242 doi 10 1007 s10529 015 1974 2 ISSN 1573 6776 PMID 26475269 S2CID 17083137 Rock Jason R Onaitis Mark W Rawlins Emma L Lu Yun Clark Cheryl P Xue Yan Randell Scott H Hogan Brigid L M 2009 08 04 Basal cells as stem cells of the mouse trachea and human airway epithelium Proceedings of the National Academy of Sciences 106 31 12771 12775 Bibcode 2009PNAS 10612771R doi 10 1073 pnas 0906850106 ISSN 0027 8424 PMC 2714281 PMID 19625615 a b Tata Purushothama Rao Rajagopal Jayaraj 2017 03 01 Plasticity in the lung making and breaking cell identity Development 144 5 755 766 doi 10 1242 dev 143784 ISSN 0950 1991 PMC 5374348 PMID 28246210 Mou Hongmei Vinarsky Vladimir Tata Purushothama Rao Brazauskas Karissa Choi Soon H Crooke Adrianne K Zhang Bing Solomon George M Turner Brett 2016 Dual SMAD Signaling Inhibition Enables Long Term Expansion of Diverse Epithelial Basal Cells Cell Stem Cell 19 2 217 231 doi 10 1016 j stem 2016 05 012 PMC 4975684 PMID 27320041 Retrieved from https en wikipedia org w index php title Air liquid interface cell culture amp oldid 1174907837, wikipedia, wiki, book, books, library,
